1. Field of the Invention
This invention relates to a method of forming tubules from diacetylenic phospholipids and to the tubule structures formed by the method. Particularly, this invention pertains to a method of forming tubules by slowly cooling multilamellar vesicles through the liquid crystalline gel point to a formation point 1-10.degree. C. below the gel phase transition temperature.
2. Description of the Prior Art
In recent years there has been an increasing interest in the use of synthetic lipid structures for a variety of applications including model membranes, immunological adjuncts, drug carriers, artificial blood substitutes and the like. The preparation of liposomes provide a practical and effective means for encapsulating liquids and solids. Liposomes are particularly useful for administrating biologically active substances into living organisms. The liposome protects substances from destruction or inactivation by bodily processes or by organisms until the substance reaches the desired reaction site in the body.
Liposomes are widely described in the literature and their general structure and methods for preparation are well known. Basically, liposomes are spheroidal structures having a lipid membrane which encapsulates materials.
Methods of preparing liposomes and encapsulates material in liposomes are described in such references as U.S. Pat. No. 4,089,801 to Schneider which discloses synthetic liposomes containing biologically active substances prepared by submitting the solvent and lipids to ultrasonic vibrations, U.S. Pat. No. 4,448,765 to Ash et al. which discloses unilamellar liposomes stabilized by the incorporation of a polymerizable lipid into the liposome membrane and U.S. Patent No. 4,133,874 to Miller et al. which discloses synthetic liposomes containing hemoglobin which functions as a blood substitute. The Miller liposomes are also prepared by ultrasonic energy. Szoka et al., provides a general review for liposome synthesis in Preparation of Unilamellar Liposomes of Intermediate Size (0.1-0.2 m) By A Combination of Reverse Phase Evaporation and Extrusion Through Polycarbonate Membranes, Biochimica et Biophysics Acta, 601 (1980) 5590571.
Since the earliest studies on phospholipids in aqueous disperson, (Bangham, A. D., M. M. Standish and J. C. Watkins, 1965, "Diffusion of Univalent Ions Across the Lamellae of Swollen Phospholipids", J. Mol. Biol. 13:238-252), pure lecithins have always been found in liposomal form, with an aqueous space contained by single or multiple continuous bilayers. This is true even for synthetic lecithins with complex thermal properties, such as dipalmitoyl phosphatidylcholine, which has at least three phase transitions (Chen, S. C., J. M. Sturtevant, and B. J. Gaffney, 1980, "Scanning Calorimetric Evidence for a Third Phase Transition in Phosphatidylcholine Bilayers", Proc. Natl, Acad. Sci. USA, 77:5060-5063). The phase transitions may change the bilayer spacings (Inoko, Y., and T. Mitsui, 1978, "Structural Parameters of Dipalmitoyl Phosphatidylcholine Lamellar Phases and Bilayer Phase Transitions", J. Physiol. Soc. Japan, 44:1918-1924), and also the surface areas of the liposomes (Yager, P., J. P. Sheridan and W. L. Peticolas, 1982, "Changes in Size and Shape of Liposomes Undergoing Chain Melting Transitions as Studied by Optical Microscopy", Biochim. Biophys. Acta. 693:485-491 ) (Evans, E. and R. Kwok, 1982, Mechanical Calorimetry of Large Dimyristoylphosphatidylcholine Vesicles in the Phase Transition Region, Biochemistry, 21:4874-4879), but the topology of the liposomes remains unchanged.
The lecithin, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC.sub.23 PC), contains a diacetylenic group halfway down each of its 23-carbon hydrocarbon chains. This lecithin polymerizes to a rigid linear polyene via a 1-4 addition reaction if irradiated with 254 nm light. Radiation, such as gamma rays or high energy electrons, will also polymerize this lecithin (Wegner, G., 1969., 1969, Topochemische Reaktionen von "Monomeren Mit Konjugierten Driefachbondungen:, Z. Naturforsch, 24b:824-832).
The polymerization reaction only proceeds when the monomers are properly aligned, as in crystals, and not in the melt (Baughman, R. H. and R. R. Chance, 1978, Fully Conjugated Polymer Crystals: Solid State Synthesis and Properties of the Polydiacetylenes, Ann. NY Acad. Sci. 313:705-725). The polymer forms only when the lipid is below its phase transition temperature (T.sub.m) of 40.degree. C. (Johnston, D. S., S. Sanghera, M. Pons, and D. Chapman, 1980, Phospholipid Polymers: Synthesis and Spectral Characteristics", Biochim, Biophys. Acta. 602:57-69; O'Brien, D. F., T. H. Whiteisdes, and R. T. Klingbiel, 1981, The Photopolymerization of Lipid-Diacetylenes in Bimolecular-layer Membranes", J. Polym, Sci. Part B. 19:95-101: Lopez, E., D. F. O'Brien and T. H. Whitesides, 1982, "Structural Effects on the Photopolymerization of Bilayer Membranes", J. Am. Chem. Soc. 104:305-307): Leaver, J., A. Alonzo, A. A. Durrani, and D. Chapman, 1983, The Physical Properties and Photopolymerization of Diacetylene-Containing Phospholipid Liposomes", Biochim. Ciophys, Acts., 732:210-218. The polymer formed from the lipid DC.sub.23 PC is dark red.
In prior work reported by the inventors using the polymerizable diacetylenic lecithin, it was found that the normal liposomes formed by gentle dispersion of the lipid above its phase transition temperature (T.sub.m) became unstable and appeared to disintegrate on cooling through the transition point or temperature (T.sub.m) (Yager, P., and P. E. Schoen, 1984, Formation of Tubules by a Polymerizable Surfactant, Mol. Cryst. Liq. Cryst. 106:371-381). The present inventors noted that the liposomes violently broke into small shards when the monomeric lipid was cooled rapidly to below 30.degree. C., but, when the lipid was cooled slowly to 37.degree. or 38.degree. C., which is within the rather broad melting transition of the compound (DC23PC), the liposomes converted quantitatively to hollow tubes over a period of a minute. The tubules were between 0.3 microns and 1 micron in diameter, with fairly thin walls, and ranged in length from a few to hundreds of micrometers.
After polymerization, the tubules do not return to liposomal form when heated. Instead, the tubules exhibit thermochromism which indicates temperature effects on the conformation of the chromophoric polymer.
While phosphatidylcholines have been considered topologically inert, other classes of lipids, such as phosphatidylethanolamines, phosphatidylglycerol, cardiolipidin, and other charged lipids can convert to nonlamellar phases, such as the inverted hexagonal (H.sub.11) phase (Cullis, P. R., and B. De Kruiff, 1979, "Lipid Polymorphism and the Functional Roles of Lipids in Biological Membranes, Biochim. Biophys, Acta, 559:399-420), or, in the case of phosphatidylserines in the presence of Ca.sup.2+, a rolled-up lamellar phase dubbed cochleate cylinders (Papahadjopoulos, D., W. J. Vaiol, K. Jacobson, and G. Poste, 1975 "Cochleate Lipid Cylinders: Formation by Fusion of Unilamellar Lipid Vesicles, Biochim. Biophys. Acta. 394:483-491).
Structures somewhat similar to tubules were reported by Leaver, J. A., Alonzo, A., A. Durrani, and D. Chapman, for a diacetylenic lecithin with 20-carbon long fatty acid chains. Leaver et al. assumed the structure to be similar to cochleate cylinders, "The Physical Properties and Photopolymerization of Diacetylene-Containing Phospholipid Liposomes," Biochim. Biophys. Acta. 732:210-218, 1983. Tubules and cochleates are superficially similar, but previously reported electron microscopic studies on DC.sub.23 PC by the inventors indicated that the tubules were somewhat different from cochleate cylinders because tubules are open ended and often consist of only a few bilayers (Yager, P. and P. E. Schoen, "Formation of Tubules by a Polymerizable Surfactant, Mol. Cryst. Liq. Cryst.," 106-371-381, 1984.
Cochleate cylinders are similar in diameter to tubules, but cochleate cylinders are not as long as tubules, and they consist of very tightly wrapped multibilayers with little or no internal aqueous space (Papahadjopoulos, D., W. J. Vail, K. Jacobson, and G. Poste, "Cochleate Lipid Cylinders; Formation by Fusion of Unilamellar Lipid Vesicles," Biochim. Biophys. Acta 394:483-491, 1975.
Recently, Nakashima, N., S. Asakuma, and T. Kunitake observed a tubular lipid structure formed from an amino-based surfactant, "Optical Microscopic Study of Helical Superstructures of Chiral Bilayer Membranes, J. Am. Chem. Soc., 107:509-510, 1985. The tubules described by Nakashima et al. are not polymerizable and so cannot be made stable or rugged. Moreover, the annealing time of these tubules is approximately a month.
The self-assembly of DC.sub.23 PC into tubules has been observed by two distinctly different pathways. Yager and Schoen, two of the inventors, previously reported one method of thermal formation of tubules which results as a consequence of cooling liquid-crystalline large multilamellar vesicles or stacked bilayers sheets through a phase transition observed at approximately 39.degree. C. "Molecular Crystals Liquid Crystals," 106:371-381, 1984.
The molecular characteristics and the polymorphic phase behavior of this lipid during the thermal formation of tubules has been characterized by vibrational spectroscopy and differential scanning calorimetry, Yager et al., Id, Rudolph et al, Biochem, Biophys. Acta, 902, 4347-359 (1987), Rudolph et al., Biophysical J. 51,185a; Burke et al., Biophysical J. 51, 185a.
Published articles suggest that fluid phase large multilamellar vesicles will form tubules directly upon cooling through the phase transition at 39.degree. . It is also believed that fluid phase small unilamellar vesicles will form tubules when supercooled to 2.degree. C., at which time they undergo a transition to a polymorphic, low temperature phase, stacked bilayer sheets.
The bilayer sheets are spectroscopically identical to the tubules. The sheets will form tubules if cycled through the transition at 39.degree. C. This indicates that one requirement for thermal formation of tubules may be a high radius of curvature i.e. greater than one micrometer. The formation of stacked bilayer sheets from highly strained small unilamellar vesicles fulfills this requirement. In addition, it has been suggested by Yager et al., Biophysical J. 49, 320a (1986) that the mechanism of thermal tubule formation occurs by the wrapping or rolling of large multilamellar vesicles. This same mechanism could apply to the formation of tubules from stacked bilayer sheets.
Another method of tubule formation has been observed by Georger et al, J. Am. Chem. Soc., 109, p 6169 (1987), and is a result of spontaneous formation in mixed solvent systems. In this method, DC.sub.23 PC is dissolved in ethanol and tubules are observed to form spontaneously upon the addition of water. The tubules formed from these two methods are morphologically similar and preliminary results on their molecular characteristics also indicate similarities.
Previous work by the inventors and their co-workers with 1,2-bis (10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC.sub.23 PC) discussed above, suggests making the tubules by slowly cooling the hydrated lipid to a point below 38.degree. C. Almost all the reported work was done with only one compound DC.sub.23 PC.
In another work, the inventors and their co-workers tried to expand the process to other 1,2 bis alkadiynoyl-sn-glycero-3-phosphocholines. These compounds were found to form tubules if the hydrated lipid is cooled to a point a few degrees below its phase transition point (T.sub.m). Singh, Price, Schnur, Schoen, and Yager, Tubule Formation by Heterobifunctional Polymerizable Lipids: Synthesis and Characterization. Abstract distributed at meeting August 1986. In another paper delivered at a meeting of the Indian Chemical Society in August of September 1986, the inventors and coworkers described work with isomers of diheptacosadiynoyl phosphocholines. An abstract was distributed at the meeting.
Suprisingly, when the slow cooling technique was applied to a broader range of diacetylenic phospholipids than just one, the process was found to be erratic. Some workers reported failure to produce tubules by the slow cooling process even with DC.sub.23 PC. The inventors work and work of others often found a high production of shards and other amorphous material when using the slow cooling process deserved in the literature DC.sub.23 PC. It has been found that this shrapnel production can be reduced by the improved methods of this invention.